Without limiting the scope of the invention, its background is described in connection with methods for the preparation of oligonucleotide “decoys”.
Oligonucleotide agents have been shown to have functional activity in vitro and thus the promise of therapeutic potential. Some of these agents are believed to operate via mechanisms such as the sequence-specific antisense translation arrest of mRNA expression or through direct binding to protein targets where they function as “decoys”. While oligonucleotide agents show therapeutic promise, various pharmacological problems must first be overcome.
Oligonucleotide agents have been used as high specificity therapeutic agents in vitro. High sensitivity to nuclease digestion, however, makes oligonucleotide agents unstable and thus impracticable for in vivo administration by either intravenous or oral routes.
From the foregoing it is apparent the there is a need in the art for methods for generating high binding, nuclease resistant oligonucleotide that retain their specifity. Also needed are compounds and methods that permit the generation of high binding, high specificity, nuclease resistant oligonucleotide agents that have an improved half-life and are target specific.
One target for oligonucleotide decoy targeting is the transcriptional activating factor NF-κB, which is activated by many factors that increase the inflammatory response. The activation of NF-κB leads to the coordinated expression of many genes that encode proteins such as cytokines, chemokines, adhesion molecules, etc., which amplify and perpetuate immune response. Because of its pivotal importance in immune function, NF-κB has been a desired target for new types of anti-inflammatory treatments. Current anti-inflammatory treatments such as glucocorticoids function at least in part through inhibition of NF-κB. Glucocorticoids, however, have endocrine and metabolic side effects when given systematically. Anti-oxidants represent another class of compounds that inhibit NF-κB activation. Currently available anti-oxidants, however, are relatively weak and have short-term effects. Aspirin and other salicylates also inhibit NF-κB, but only at relatively high concentrations that may have undesirable side effects. Naturally occurring inhibitors of NF-κB such as gliotoxin are potent and relatively specific, but also may have toxic effects.
NF-κB may have a particularly important role in the genesis of endotoxic shock, a disease entity of major clinical importance. In endotoxic shock, a series of intracellular signaling events, in which NF-κB activation figures importantly, lead to enhanced transcription of a variety of proinflammatory mediator genes, including tumor necrosis factor α, interleukin-1, inducible nitric oxide synthetase. These secreted mediators in turn lead to increased adhesion molecule expression on leukocytes and endothelial cells, increased tissue factor expression on monocytes and endothelial cells, promoting coagulation, vasodilation, capillary leakiness, and myocardial suppression. (Murphy, et al., New Horizons (1998) 6:181). Strong support for the role of NF-κB in septic shock in humans is afforded by the recent demonstration that sustained, increased NF-κB binding activity in nuclei of peripheral blood monocytes from septic patients predicted mortality.
From the foregoing it is apparent the there is a need in the art for methods for generating high binding, nuclease resistant aptamers that retained their specificity.